The rabies virus is a non-segmented, single-stranded, linear RNA rhabdovirus or lyssavirus with negative sense polarity of the family Rhabdoviridae, which has a bullet-like shape having one round or conical end while the other end is planar or concave in shape. The round or conical end of the virion possesses a lipoprotein envelope with knob-like spikes composed of glycoprotein G. In addition to glycoprotein G, the lipid membrane or viral envelope surrounding the core structure has a second, inner layer consisting of a matrix protein (M). The external surface glycoprotein G is responsible for cell attachment and has been identified as the antigenic substance that is responsible for the virulence or pathogenicity of the rabies virus as well as the host immune response.
With few exceptions, rabies invariably results in fatal neurological disease in humans and animals, and remains a serious global public health concern. The majority of human deaths stemming from rabies have occurred in Africa, Asia and South America but a rabies epidemic has also recently become problematic in the United States due to a rapidly growing population of infected raccoons. Other primary virus carriers of concern are the skunk, largely in the mid-western states, and bats, the main source for most human cases in the U.S. In addition to the infected wildlife such as the raccoons, skunks, foxes, wolves, etc., humans typically become infected with rabies through the bite of infected dogs and cats. Dogs continue to be the main hosts of the rabies virus in Africa and Asia where canine rabies is endemic and are still responsible for most of the human deaths that occur from rabies worldwide. It is of particular importance to mankind, therefore, to prevent the rabies virus infection in domestic pets such as dogs, cats and ferrets.
Louis Pasteur and Emile Roux developed the first rabies vaccination in 1885. The early nerve tissue-derived vaccine consisted of a virus sample taken from infected rabbits and dried to weaken its pathogenicity. Some developing countries still use comparable neural tissue rabies vaccines and, although much less expensive than modern cell culture vaccines, they are not nearly as effective and carry a significant risk of neurological side effects.
In 1967, the human diploid cell rabies vaccines (HDCV) were developed for human vaccination using the attenuated Pitman-Moore L503 strain of the virus. Now available on the market are less expensive, highly purified chick embryo culture vaccine (PCEC) and purified Vero cell rabies vaccine. The latter Vero cell culture vaccine uses the attenuated Wistar strain of the rabies virus while the Vero cell line is its host. Despite the attenuation, the Vero cell culture vaccine has the potential to revert to virulence. Consequently, preparation of the rabies vaccine requires extreme care by the workers to avoid the accidental dissemination of viral infections with the strain as a result of a rabies virus surviving the inactivation process. The PCEC vaccine has the added disadvantage that it cannot be given to anyone with an allergy to eggs or chickens. Additional difficulties handling, manufacturing or using live rabies virus in vaccines are well known to those in the pharmaceutical and veterinary arts.
In view of the disadvantages of conventional rabies viral vaccines, research has been aimed at the use of recombinant raccoon poxviruses and the insertion of foreign genes such as the rabies virus glycoprotein genes into the thymidine kinase (tk) locus of raccoon poxviruses. It was found that certain early constructs of recombinant raccoon poxviruses could express the foreign viral antigenic substance or foreign DNA and elicit host protective immune responses in other animals such as dogs and cats. However, the construction of safe and effective recombinant raccoon poxviruses to be useful as vaccines is a complex matter and involves many factors that must be taken into consideration. In particular, the ability to create recombinant raccoon poxviruses varying the position of the exogenous or foreign genes inserted into different regions beyond the known tk locus as well as the particular selection of functional foreign antigenic DNA fragments require significant experimental studies to determine the stability, the safety and the efficacy of the resultant recombinant poxviruses. Several attempts, therefore, to obtain a commercially viable recombinant raccoon poxvirus vaccine have been the focus of numerous efforts in the veterinary vaccine field.
As such, there is a significant amount of published information on the topic of recombinant raccoon poxvirus as vaccines. More recently, for instance, an oral genetically recombined virus vaccine for raccoon rabies has been described in U.S. Patent Application No. 2005/0282210. A gene that produces a protein in the rabies viral outer coat was inserted into a live vaccinia virus using recombinant DNA technology. When the modified vaccinia virus infects a normal animal, it produces the antigenic protein normally made by the rabies virus. The biological system of the victim recognizes the protein as foreign; and the animal develops active immunity. Specifically, Patent Application No. 2005/0282210 is drawn to a method of eliciting an immune response in a skunk or mongoose which involves administering a composition that consists of a viral vector comprising a rabies surface glycoprotein gene that has been inserted into the viral vector genome of the vaccinia. The disclosure suggests the potential insertion sites for the polynucleotide or polynucleotides to be expressed at the thymidine kinase (tk) gene or insertion site, the hemagglutinin (ha) gene or insertion site, or the region encoding the inclusion body of the A type (ATI) of a vaccinia virus; ORF(s) C3, C5 and/or C6 in the case of canarypox virus; ORFs F7 and/or F8 in the case of fowipox virus but the document only exemplifies the use of the vaccinia virus vector in which the rabies glycoprotein G is derived from the ERA strain and is inserted only in the tk site of the vaccinia.
U.S. Pat. No. 7,074,413 discloses the design of recombinant rabies virus vaccines by replacing the glycoprotein of a non-neuroinvasive rabies strain with that of a street or neuroinvasive rabies virus to produce an attenuated recombinant rabies virus for vaccination or constructing a recombinant rabies virus expressing a pro-apoptotic protein.
U.S. Pat. No. 6,719,981 shows attenuated rabies virus mutants and live attenuated anti-rabies vaccines comprising said mutants in which the recombinant rabies virus mutant of a Street Alabama Dufferin strain (SAD D29) comprises a mutation in the G protein of the viral genome, wherein said mutation comprises a particular substitution of a AGA codon encoding Arg333 with a GAC codon.
U.S. Pat. No. 6,294,176 relates to a recombinant raccoon poxvirus vaccine that consists of a raccoon poxvirus viral genome which contains a foreign DNA sequence inserted into a non-essential region within the HindIII “U” genomic region, the HindIII “M” genomic region or the HindIII “N” genomic region of the raccoon poxvirus genome. The raccoon poxvirus viral genome is described in the patent as containing a deletion in the raccoon poxvirus host range gene of the viral genome. The patent provides a homology vector for producing the recombinant raccoon poxvirus by inserting the foreign DNA sequence into the raccoon poxvirus genome. While there is the broad suggestion that the recombinant raccoon poxvirus may contain foreign DNA encoding an antigenic polypeptide from the rabies virus, there is no exemplification relevant thereto. In addition, DNA sequence analysis indicates that the HindIII “U” genomic region disclosed by the patentees is not the hemagglutinin (ha) insertion and/or the thymidine kinase (tk) regions of the recombinant raccoon poxvirus genome.
U.S. Pat. No. 6,241,989 and its continuation U.S. Pat. No. 7,087,234 deal with multivalent recombinant raccoon poxviruses, containing more than one exogenous gene inserted into either the thymidine kinase gene or the hemagglutinin gene. Disclosed in these patents is the use of the multivalent recombinant raccoon poxviruses as vaccines to immunize felines against feline pathogens. Also disclosed is a method of making a multivalent recombinant raccoon poxvirus by a recombinant process involving the construction of an insertion vector into which the exogenous genes are inserted; and flanking the inserted genes are sequences which can recombine into the raccoon poxvirus thymidine kinase gene or the hemagglutinin gene; introducing both the insertion vector containing the exogenous genes, and raccoon poxvirus into susceptible host cells; and selecting the recombinant raccoon poxvirus from the resultant plaques. The multivalent, recombinant raccoon poxvirus of the patents can infect and replicate in feline cells, and contains more than one exogenous gene inserted into a region consisting of a hemagglutinin gene or a thymidine kinase gene of the raccoon poxvirus genome which is non-essential for viral replication and each exogenous gene encodes a feline pathogen antigen. The patents describe exogenous genes encoding feline pathogen antigens such as feline leukemia virus (FeLV Env), feline immunodeficiency virus (FIV Gag), feline immunodeficiency virus (FIV Env), feline infectious peritonitis virus (FIPV M), feline infectious peritonitis virus (FIPV N), feline calicivirus (FCV capsid protein), feline panleukopenia virus (FPV VP2) and rabies-G.
Although U.S. Pat. Nos. 6,241,989 and 7,087,234 suggest that both the thymidine kinase (tk) gene and the hemagglutinin (ha) gene of the raccoon poxvirus genome can be used for insertion of exogenous genes by recombination, the specific examples only show how to generate a multivalent RCNV-based recombinant FPV VP2 and rabies virus (RCNV/FPV/RAB-G) by homologous recombination of the flanked vaccinia virus tk gene sequence and a separate homologous recombinant raccoon poxvirus containing FCV capsid protein gene inserted into the ha gene. There are no claims or exemplification teaching how to construct and/or use a recombinant raccoon poxvirus containing multiple foreign antigenic material on both thymidine kinase and the hemagglutinin genes. Moreover, the patents do not teach or disclose any recombinant constructs having the rabies virus glycoprotein gene inserted into the ha site of the raccoon poxvirus genome.
U.S. Pat. No. 6,106,841 relates to a method for immunizing an animal against a heterologous antigen. The method describes administering to the animal via the conjunctival route, a composition comprising a recombinant raccoon poxvirus having a nucleic acid molecule encoding the heterologous antigen. Antigens are described as calicivirus, coronavirus, herpesvirus, immunodeficiency virus, infectious peritonitis virus, leukemia virus, parvovirus antigen, rabies virus, Bartonella, Yersinia, Dirofilaria, Toxoplasma, flea antigen or flea allergen, midge antigen or allergen, mite antigen or allergen and a tumor antigen. The patent further discloses a recombinant raccoon poxvirus that comprises a nucleic acid molecule encoding an immunomodulator such as granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), interleukins, interferon gamma and the like. Also described therein are methods using a recombinant raccoon poxvirus genome having a heterologous nucleic acid molecule in the raccoon poxvirus gene selected from thymidine kinase, hemagglutinin, serpin, cytokine receptor and interferon receptor genes in which the heterologous nucleic acid molecule is operatively linked to a transcription control sequence consisting of a p11 poxvirus promoter, a p7.5 poxvirus promoter or a synthetic poxvirus promoter.
U.S. Pat. No. 6,024,953 describes a vaccinia virus that contains all or part of a DNA sequence coding for an antigenic glycoprotein of rabies. In particular, the patent discloses a hybrid vaccinia virus containing a DNA sequence which encodes the amino acid sequence of rabies glycoprotein G that has been inserted in a vaccinia thymidine kinase (tk) gene under the control of the 7.5 K vaccinia virus promoter and a vaccine for preventing and treating rabies that consists of the hybrid vaccinia virus and a pharmaceutically acceptable carrier.
U.S. Pat. No. 5,348,741 deals with a plasmid vector that has been constructed with recombinant DNA comprising a rabies virus glycoprotein G gene operatively linked to a vaccinia P11 late promoter. The rabies virus glycoprotein G gene is derived from Challenge Virus Standard strain. The gene and the promoter are inserted into the thymidine kinase (tk) locus of the vaccinia or cowpox virus vector. The patent states that the recombinant vaccinia virus expresses the gene for rabies virus glycoprotein in cells and induces production of glycoprotein for immunization against rabies. The patent indicates that the recombinant virus could be applied for the production of anti-rabies vaccine and of G antigen antibody and related immunological reagents for research or diagnostic purposes. Notably, the patent does not describe inserting the rabies virus glycoprotein G gene into any other site besides the tk locus and does not give any suggestion of using a raccoon poxvirus as the vector.
An early DNA rabies vaccine is described in U.S. Pat. No. 5,830,477, which relates to a vaccinia virus that contains all or part of a DNA sequence coding for an antigenic glycoprotein of rabies. The patent concerns an oral vaccine for preventing or treating rabies in a mammal consisting of a hybrid vaccinia virus that contains and expresses a DNA sequence encoding the amino acid sequence rabies glycoprotein G wherein the DNA sequence is present in a non-essential segment of the vaccinia virus and a pharmaceutically acceptable carrier. Specifically, the patent only exemplifies a hybrid vaccinia virus vaccine in which the rabies glycoprotein G is under the control of a 7.5K vaccinia promoter and is present in the vaccinia thymidine kinase (tk) gene.
U.S. Pat. No. 5,266,313 relates to the use of raccoon poxvirus as a substrate for insertion and expression of the nucleotide coding sequence of heterologous organisms. The patent describes the production of two infectious raccoon poxvirus recombinants for expressing rabies virus surface spike glycoprotein (G) by homologous recombination between raccoon poxvirus DNA and chimeric plasmids used for production of vaccinia virus recombinants by thymidine kinase (tk) insertional inactivation.
Also of interest as background material to the present invention are previous patents relating to the common use of promoters, namely, those sequences that positively regulate the transcription of a gene such as the promoters used with the foreign gene protein coding sequence that comprises the foreign gene or exogenous gene material that is being inserted and expressed by a recombinant vector. For example, U.S. Pat. No. 6,998,252 concerns recombinant poxviruses, such as vaccinia, that comprise a first DNA sequence encoding a polypeptide that is foreign to poxvirus and a poxvirus transcriptional regulatory sequence, wherein the transcriptional regulatory sequence is adjacent to and exerts transcriptional control over the DNA sequence and the segment is positioned within a nonessential genomic region of the recombinant poxvirus. Construction of plasmids containing the promoter from the 7.5K polypeptide gene (7.5K gene) of vaccinia virus, the restriction endonuclease sites for insertion of foreign protein coding sequences and the interrupted vaccinia virus thymidine kinase (tk) gene as flanking DNA are shown.
U.S. Pat. No. 7,045,313 similarly relates to methods and compositions for the use of vaccinia virus or other poxviruses as vectors for expression of foreign genes in which the expression is obtained by combining the vaccinia virus transcriptional regulatory sequence with uninterrupted foreign protein coding sequences in vitro to form a chimeric gene. The patent shows how the chimeric gene is flanked by DNA from a non-essential region of the vaccinia virus genome to provide sites for in vivo homologous recombination. These steps are facilitated in the patent disclosure by the construction of plasmids that contain multiple restriction endonuclease sites, next to the vaccinia transcriptional regulatory sequences, for insertion of the foreign protein coding sequence. The patent further shows the plasmids comprising a segment comprised of a first DNA sequence encoding a polypeptide that is foreign to vaccinia virus and a vaccinia virus promoter sequence, wherein said promoter sequence is adjacent to and exerts transcriptional control over said first DNA sequence; and, flanking said segment, DNA from a nonessential region of a vaccinia genome. Specifically, the promoter sequence is one that in vaccinia virus regulates the thymidine kinase (tk) gene or a vaccinia gene encoding a 7.5 K polypeptide. Transfection procedures are illustrated therein to introduce the DNA into cells where homologous recombination results in the insertion of the chimeric gene into a non-essential region of the vaccinia virus genome.
Also relevant to standard promoters known in the art, U.S. Pat. No. 7,208,313 relates to vaccinia virus expression vectors produced with a negative thymidine kinase (tk) phenotype and a negative vaccinia virus growth factor phenotype. The foreign gene is placed under the control of a vaccinia virus promoter and integrated into the genome of the mutant vaccinia virus. Alternatively, expression can be achieved by transfecting a shuffle vector or plasmid containing the vaccinia promoter-controlled gene into a cell that has been infected with vaccinia virus and introducing the exogenous sequence by homologous recombination.
Synthetic early-late vaccinia virus promoters are illustrated in U.S. Pat. Nos. 6,183,750; 7,067,248 and others. U.S. Pat. No. 6,183,750 describes a recombinant poxvirus, such as vaccinia virus, fowipox virus or canarypox virus, containing foreign DNA from herpesvirus and a promoter operably linked to the DNA for expressing the DNA, wherein the promoter is superimposed on or ligated to the initiation codon of the DNA. The promoter-gene linkage is positioned in the plasmid construct so that the linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. A condition for expression of inserted DNA is the presence of the promoter in the proper relationship to the inserted DNA, that is, the promoter should be placed so that it is located upstream from the DNA sequence to be expressed. U.S. Pat. No. 7,067,248 additionally shows ways to use a synthetic early-late promoter to express a substrate protein from vaccinia virus. See also S. Chakrabarti et al., “Compact, Synthetic, vaccinia virus early/late promoter for protein expression,” BioTechniques 23: 1094-1097 (1997).
Other material relevant to the background of the present invention can be found in the following literature citations: A. D. Alexander et al, “Survey of wild mammals in a Chesapeake Bay area for selected zoonoses,” J. Wildlife Dis. 8: 119-126 (1972); C. Bahloul et al., “DNA-based immunization for exploring the enlargement of immunological cross reactivity against the lyssaviruses,” Vaccine 16: 417-425 (1998); S. Chakrabarti et al., “Compact, Synthetic, vaccinia virus early/late promoter for protein expression,” BioTechniques 23: 1094-1097 (1997); J. C. DeMartini et al., “Raccoon poxvirus rabies virus glycoprotein recombinant vaccine in sheep,” Arch. Virol. 133: 211-222 (1993); B. Dietzschold et al., “Rhabdoviruses,” In: Fields Viology, ed. B. N. Fields et al., 3rd edition, New York: Raven Press, pp. 1137-1159 (1996); J. J. Esposito et al., “Vaccinia virus recombinants expressing rabiesvirus glycoprotein protect against rabies,” Virus Genes 1: 7-21 (1987); J. J. Esposito et al., “Successful oral rabies vaccination of raccoon poxvirus recombinants expressing rabies virus glycoprotein” Virology 165: 313-316 (1988); J. J. Esposito, “Live poxvirus-vectored vaccines in wildlife immunization programmes: the rabies paradigm. Res. Virol. 140: 480-482 (1989); J. J. Esposito et al., “Oral Immunization of Animals with Raccoon Poxvirus Expressing Rabies Virus Glycoprotein,” In: Vaccines 89, eds. R. Lerner, H. Ginsberg, R. M. Chanock and F. Brown, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 403-408 (1989); J. J. Esposito et al., “Raccoon Poxvirus Rabies-Glycoprotein Recombinant Oral Vaccine for Wildlife: Further Efficacy and Safety Studies and Serosurvey for Raccoon Poxvirus,” In: Vaccines 92, eds. F. Brown, R. M. Chanock, H. S. Ginsberg and R. A. Lerner, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 321-329 (1992); Y. F. Herman, “Isolation and characterization of a naturally occurring pox virus of raccoons,” In: Bacteriol. Proc., 64th Annual Meeting of the American Society for Microbiology, p. 117 (1964); L. Hu et al., “Raccoon poxvirus live recombinant feline panleukopenia virus VP2 and rabies virus glycoprotein bivalent vaccine,” Vaccine 15: 1466-1472 (1996); L. Hu et al., “Raccoon poxvirus feline panleukopenia virus VP2 recombinant protects cats against FPV challenge,” Virology 218:248-252 (1997); R. I. Macfarlan et al., “Stimulation of cytotoxic T-lymphocyte responses by rabies virus glycoprotein and identification of an immunodominant domain,” Mol. Immunol. 23: 733-741 (1986); J. E. Osorio et al., “Recombinant raccoon pox vaccine protests mice against lethal plaque,” Vaccine 21: 1232-1238 (2003); P. Perrin et al., “Rabies immunosome (subunit vaccine) structure and immunogenicity. Pre- and post-exposure protection studies,” Vaccine 3: 325-332 (1985); S. J. Spatz et al., “Immunological characterization of the feline herpesvirus-1 glycoprotein B and analysis of its deduced amino acid sequence,” Virology 197: 125-136 (1993); S. J. Spatz et al., “Identification of the feline herpesvirus type 1 (FHV-1) genes encoding glycoproteins G, D, I and E: expression of FHV-1 glycoprotein D in vaccinia and raccoon poxviruses,” J. Gen. Virol. 75: 1235-1244 (1994); J. Taylor et al., “Efficacy studies on a canarypox-rabies recombinant virus,” Vaccine 9: 190-192 (1991); E. K. Thomas et al., “Further characterization of raccoonpox virus,” Arch. Virol. 49:217-227 (1975); E. Yelverton et al., “Rabies virus glycoprotein analogs: Biosynthesis in Escherichia coli,” Science 219: 614-620 (1983).
Compared to conventional inactivated rabies vaccines, successful development of a safe and effective, adjuvant-free raccoon poxvirus-vectored rabies vaccine would result in important advantages in avoiding adjuvant-associated sarcoma side effects in cats, ensuring employee safety during vaccine production and completely eliminating any chance of rabies virus surviving inactivation and decontamination procedures used in commercial production. An art-recognized need still exists for a safe and effective rabies virus vaccine for household pets that would adequately protect the pets from being infected with the rabies virus and, in turn, protect their human owners from fatal infections. Also needed is a viable method for the prevention of rabies and the amelioration of harmful neurological effects in mammals.
The foregoing objects are accomplished by providing a safe and efficacious recombinant rabies vaccine that results in long-lasting immunity in dogs and cats in the form of the new raccoon poxvirus vector construct of a rabies vaccine as described herein.
All patents and publications cited in this specification are hereby incorporated by reference in their entirety.